Specific Aims. Our goal is use integrated structural, chemical and molecular systems biology approaches to develop estrogen receptor (ER) antagonists with improved efficacy in targeting distinct mechanisms of hormone-resistant breast cancer, enabling more personalized medicine. As 70-80% of women present with ER? positive disease and up to 50% fail on endocrine therapies with disease recurrence, most deaths are of ER?-positive patients, highlighting a significant unmet clinical need for improved therapies. Importantly, patients who fail on one hormone therapy typically respond to a different one having an alternate mechanism of action. Aim 1. Use structural and chemical systems biology approaches to identify rules for antagonizing ER? in the context of different ER? -based mechanisms of resistance, and as co-treatments with other non-ER? resistance targeting agents. These approaches aim to overcome resistance and synergize with ER? antagonists in ER(+) breast cancer. We have developed a systems biology approach enabling crystallization of the ER? ligand-binding domain in parallel with many ligands. Analysis of dozens of crystal structures in parallel allows us to implement an unbiased approach to identify subtle structural perturbations in the sub- range (within the noise of the individual structures) that contribute significantly to ER?-regulated proliferation, which we call super-resolution x-ray crystallography. We will use this approach to identify structural features that drive transcriptional repression, receptor degradation, and therapeutic efficacy of structurally diverse ligands in different resistance models, and in synergy studies with co-treatment regimens, including PI3K, mTOR, and CDK4/6 inhibitors. We have developed a quantitative high-throughput screening assay for tracking receptor degradation. Key ER?-driven target genes, coregulator interaction and quantitative degradation assays will be used to mechanistically tie receptor structure to anti- proliferative effects in treatment-sensitive and resistant breast cancer models, including constitutively active mutant ER?, hyperactive growth factor signaling, and overexpression of SRC3/AIB1, in a chemical systems biology approach that we call ligand class analysis. Aim 2. Design and synthesize ER? full antagonists with novel pharmacophores that produce distinct structural perturbations of the receptor, and characterize their in vivo pharmacology in animal models of treatment-resistant ER?(+) breast cancer. The adamantyl scaffold provides a stable core with which we will explore how different, novel side chains perturb ER? structure to mediate transcriptional repression and overcome resistance. Because we found that the alternative OBHS-N core compounds produced full ER antagonism and degradation without side chains, by acting as indirect antagonists through perturbations within the pocket, this OBHS-N core now provides a novel, isosteric platform for adding side chains to access distinct and combined direct and indirect mechanisms of antagonism. We will optically barcode a number of resistance models, allowing for a multiplexed in vivo assessment of ligand efficacy. With both classes, we will evaluate them against wild type and tamoxifen-resistant ER? (+) models in vivo. On-target activity will be verified with ER? knockout and ER-negative breast cancer cells as control, with validation of effects on key ER? transcripts in vivo.